2.1. Derivatives of Methotrexate and Aminopterin
Rosowsky et al. (1981, J. Med. Chem 24: 1450, hereinafter "Rosowsky et al. I") describes a method for preparing hydrazide derivatives of methotrexate by coupling 4-amino-4-deoxy-N.sup.10 -methylpteroic acid (MAPA) to an appropriately protected L-glutamic acid precursor coupled by means of a peptide bond forming agent such as diethyl phosphorocyanidate followed by reaction with hydrazine and removal of the protective ester moiety. The MAPA can be prepared by known chemical synthetic methods [see e.g., Rosowsky et al., 1985, J. Med. Chem. (hereinafter "Rosowsky et al. II"); Chaykovsky et al., 1974, J. Med. Chem 17: 1212; Piper et al., 1977, J. Org. Chem. 42: 208] or by cleavage of methotrexate with carboxypeptidase G.sub.1 (see Martinelli et al., 1979, J. Med. Chem. 22: 869; McCullough et al., 1971, J. Biol. Chem. 246: 720). According to the method of Rosowsky et al., I supra, L-glutamic acid-.gamma.-methyl ester is esterified with t-butylacetate in the presence of 70% perchloric acid to form .alpha.-t-butyl-.gamma.-methyl glutamic acid. The C-protected .alpha.-t-butyl-.gamma.-methyl glutamic acid ester is condensed with MAPA, by heating MAPA with diethylphosphorocyanidate at about 80.degree. C. for 2 minutes in dimethylformamide (DMF) containing triethylamine, then adding the glutamic acid derivative. Heating is continued at about 80.degree. C. for another 2 hours, followed by solvent evaporation under reduced pressure and column chromatography on silica gel forming MTX-.alpha.-t-butyl-.gamma.-methyl ester. Reaction of the MTX-diester with hydrazine in methanol solution at 4.degree. C. for 60 hours yields the .alpha.-butyl ester .gamma.-hydrazide. Acidolysis of the butyl ester in 1 N HCl at 50.degree. C. for 1 hour, followed by ion-exchange chromatography on DEAE-cellulose forms the desired MTX-.gamma.-hydrazide.
The method of Rosowsky et al. has a number of disadvantages including:
(1) the intermediate used, for example, .alpha.-methyl ester-t-butyl glutamic acid is unstable as a free base, and
(2) significant yields of aminopterin-.alpha.-hydrazide could not be obtained using this method which utilizes the readily available MAPA analog 4-amino-4-deoxy-N.sup.10 -formyl pteroic acid because undesirable premature removal of the formyl protecting group from 4-amino-4-deoxy-N.sup.10 -formyl pteroic acid would occur during subsequent hydrazinoysis required to form the corresponding aminopterin analog.
A number of amino acid and peptide derivatives of methotrexate and/or aminopterin have been described. See, for example, Rosowsky, U.S. Pat. No. 4,490,529 entitled "Cysteic Acid and Homocysteic Acid Analogues of Methotrexate and Aminopterin"; Kempton et al, 1982, J. Med. Chem 25:475: Piper et al., 1982, J. Med. Chem. 25:182.
None of these references either describes or suggests that a reactive amine moiety of the attached amino acid or peptide might be useful for coupling the methotrexate or aminopterin analog to an antibody molecule via an oxidized carbohydrate moiety of the antibody.